Utilization of Nitrogen-Doped Graphene Quantum Dots to Neutralize ROS and Modulate Intracellular Antioxidant Pathways to Improve Dry Eye Disease Therapy.

Purpose: Patients afflicted with dry eye disease (DED) experience significant discomfort. The underlying cause of DED is the excessive accumulation of ROS on the ocular surface. Here, we investigated the nitrogen doped-graphene quantum dots (NGQDs), known for their ROS-scavenging capabilities, as a treatment for DED. Methods: NGQDs were prepared by using citric acid and urea as precursors through hydrothermal method. The antioxidant abilities of NGQDs were evaluated through: scavenging the ROS both extracellular and intracellular, regulating the nuclear factor-erythroid 2-related factor (Nrf2) antioxidant pathway of human corneal epithelial cells (HCECs) and their transcription of inflammation related genes. Furthermore, NGQDs were modified by Arg-Gly-Asp-Ser (RGDS) peptides to obtain RGDS@NGQDs. In vivo, both the NGQDs and RGDS@NGQDs were suspended in 0.1% Pluronic F127 (w/v) and delivered as eye drops in the scopolamine hydrobromide-induced DED mouse model. Preclinical efficacy was compared to the healthy and DPBS treated DED mice. Results: These NGQDs demonstrated pronounced antioxidant properties, efficiently neutralizing free radicals and activating the intracellular Nrf2 pathway. In vitro studies revealed that treatment of H2O2-exposed HCECs with NGQDs induced a preservation in cell viability. Additionally, there was a reduction in the transcription of inflammation-associated genes. To prolong the corneal residence time of NGQDs, they were further modified with RGDS peptides and suspended in 0.1% Pluronic F127 (w/v) to create RGDS@NGQDs F127 eye drops. RGDS@NGQDs exhibited superior intracellular antioxidant activity even at low concentrations (10 µg/mL). Subsequent in vivo studies revealed that RGDS@NGQDs F127 eye drops notably mitigated the symptoms of DED mouse model, primarily by reducing ocular ROS levels. Conclusion: Our findings underscore the enhanced antioxidant benefits achieved by modifying GQDs through nitrogen doping and RGDS peptide tethering. Importantly, in a mouse model, our novel eye drops formulation effectively ameliorated DED symptoms, thereby representing a novel therapeutic pathway for DED management.

A tissue-adhesive F127 hydrogel delivers antioxidative copper-selenide nanoparticles for the treatment of dry eye disease.

Dry eye disease (DED) is currently the most prevalent condition seen in ophthalmology outpatient clinics, representing a significant public health issue. The onset and progression of DED are closely associated with oxidative stress-induced inflammation and damage. To address this, an aldehyde-functionalized F127 (AF127) hydrogel eye drop delivering multifunctional antioxidant Cu2-xSe nanoparticles (Cu2-xSe NPs) was designed. The research findings revealed that the Cu2-xSe nanoparticles exhibit unexpected capabilities in acting as superoxide dismutase and glutathione peroxidase. Additionally, Cu2-xSe NPs possess remarkable efficacy in scavenging reactive oxygen species (ROS) and mitigating oxidative damage. Cu2-xSe NPs displayed promising therapeutic effects in a mouse model of dry eye. Detailed investigation revealed that the nanoparticles exert antioxidant, anti-apoptotic, and inflammation-mitigating effects by modulating the NRF2 and p38 MAPK signalling pathways. The AF127 hydrogel eye drops exhibit good adherence to the ocular surface through the formation of Schiff-base bonds. These findings suggest that incorporating antioxidant Cu2-xSe nanoparticles into a tissue-adhesive hydrogel could present a highly effective therapeutic strategy for treating dry eye disease and other disorders associated with reactive oxygen species. STATEMENT OF SIGNIFICANCE: A new formulation for therapeutic eye drops to be used in the treatment of dry eye disease (DED) was developed. The formulation combines copper-selenium nanoparticles (Cu2-xSe NPs) with aldehyde-functionalized Pluronic F127 (AF127). This is the first study to directly examine the effects of Cu2-xSe NPs in ophthalmology. The NPs exhibited antioxidant capabilities and enzyme-like properties. They effectively eliminated reactive oxygen species (ROS) and inhibited apoptosis through the NRF2 and p38 MAPK signalling pathways. Additionally, the AF127 hydrogel enhanced tissue adhesion by forming Schiff-base links. In mouse model of DED, the Cu2-xSe NPs@AF127 eye drops demonstrated remarkable efficacy in alleviating symptoms of DED. These findings indicate the potential of Cu2-xSe NPs as a readily available and user-friendly medication for the management of DED.

Biocompatible tumor-targeted GQDs nanocatalyst for chemodynamic tumor therapy.

To deal with the complex tumor microenvironment (TME), chemodynamic therapy (CDT) has been developed, which uses nanocatalysts simulating peroxidase to convert high concentration hydrogen peroxide (H2O2) into highly toxic hydroxyl radicals (˙OH) in situ and effectively kills tumor cells. Due to the low catalytic activity of traditional nanocatalysts, the present CDT treatment has to be combined with other anti-tumor therapies, which increases the complexity and uncertainty of the treatment. Thus, developing new nanocatalysts with stable and high enzymatic activity is the key point to CDT treatment. Graphene quantum dots (GQDs) are important metal-free catalysts with intrinsic peroxidase-like activity due to their excellent electron transport performance. Here, we prepare a nitrogen-doped GQD (NGOD) nanocatalyst, which displays much higher peroxidase activity than known metal nanocatalysts. The NGQD nanocatalyst is further grafted with RGDS peptide-modified polyethylene glycol (PEG), which guides the nanocatalyst to the tumor area and increases its circulation time in blood. The as-produced RGDS-PEG@NG nanocatalyst displays stable and high peroxidase activity, which achieves the conversion of H2O2 → ˙OH in the TME. Through an in vivo study it has been observed that RGDS-PEG@NGs obviously inhibit tumor growth without combining with other treatment methods and show excellent biocompatibility, which provides a unique idea for the application of GQDs in CDT.

Mechanical Properties of Lightweight Cementitious Cellular Composites Incorporating Micro-Encapsulated Phase Change Material.

This work focuses on combining digitally architected cellular structures with cementitious mortar incorporating micro-encapsulated phase change material (mPCM) to fabricated lightweight cementitious cellular composites (LCCCs). Voronoi structures with different randomness are designed for the LCCCs. Aided by the indirect 3D printing technique, the LCCCs were prepared with a reference mortar (REF) and a mortar incorporating mPCM. The compressive behavior of the LCCCs was studied at the age of 28 days, by experimental and numerical methods. It was found that the highly randomized Voronoi structure and the mPCM have minor negative influence on the compressive properties of the LCCCs. The mPCM incorporated LCCCs have high relative compressive strength compared to conventional foam concrete. Furthermore, the critical role of air voids defects on the compressive behavior was identified. The highly randomized porous Voronoi structure, high mPCM content and good compressive strength ensure the LCCCs' great potential as a novel thermal insulation construction material.

Oxygen content-related DNA damage of graphene oxide on human retinal pigment epithelium cells.

Arguments regarding the biocompatibility of graphene-based materials (GBMs) have never ceased. Particularly, the genotoxicity (e.g., DNA damage) of GBMs has been considered the greatest risk to healthy cells. Detailed genotoxicity studies of GBMs are necessary and essential. Herein, we present our recent studies on the genotoxicity of most widely used GBMs such as graphene oxide (GO) and the chemically reduced graphene oxide (RGO) toward human retinal pigment epithelium (RPE) cells. The genotoxicity of GO and RGOs against ARPE-19 (a typical RPE cell line) cells was investigated using the alkaline comet assay, the expression level of phosphorylated p53 determined via Western blots, and the release level of reactive oxygen species (ROS). Our results suggested that both GO and RGOs induced ROS-dependent DNA damage. However, the DNA damage was enhanced following the reduction of the saturated C-O bonds in GO, suggesting that surface oxygen-containing groups played essential roles in the reduced genotoxicity of graphene and had the potential possibility to reduce the toxicity of GBMs via chemical modification.

Biocompatible nucleus-targeted graphene quantum dots for selective killing of cancer cells via DNA damage.

Graphene quantum dots (GQDs) are nano-sized graphene slices. With their small size, lamellar and aromatic-ring structure, GQDs tend to enter into the cell nucleus and interfere with DNA activity. Thus, GQD alone is expected to be an anticancer reagent. Herein, we developed GQDs that suppress the growth of tumor by selectively damaging the DNA of cancer cells. The amine-functionalized GQDs were modified with nucleus targeting TAT peptides (TAT-NGs) and further grafted with cancer-cell-targeting folic acid (FA) modified PEG via disulfide linkage (FAPEG-TNGs). The resulting FAPEG-TNGs exhibited good biocompatibility, nucleus uptake, and cancer cell targeting. They adsorb on DNA via the π-π and electrostatic interactions, which induce the DNA damage, the upregulation of the cell apoptosis related proteins, and the suppression of cancer cell growth, ultimately. This work presents a rational design of GQDs that induce the DNA damage to realize high therapeutic performance, leading to a distinct chemotherapy strategy for targeted tumor therapy.

[Risk of anticoagulation therapy in surgical intensive care unit patients predicted by thromboelastograph].

OBJECTIVE: To explore the correlation between thromboelastography (TEG) parameters and the risk of venous thromboembolism (VTE) and bleeding in patients receiving anticoagulant therapy in surgical intensive care unit (SICU). METHODS: 205 patients received low molecular weight heparin (LMWH) anticoagulant therapy admitted to SICU of Tianjin Hospital from December 2016 to December 2017 were consecutively enrolled. TEG detection was performed in all patients at 1 day after anticoagulation therapy, and coagulation reaction time (R value), blood clot generation time (K value), blood clot generation rate (α angle) and maximum width value (MA value) were recorded. At the same time, the traditional coagulation function test was carried out, and prothrombin time (PT), activated partial thromboplastin time (APTT) and D-dimer levels were also recorded. The incidence of deep venous thrombosis (DVT), pulmonary embolism (PE) and bleeding during hospitalization were observed. Multivariate Logistic regression analysis was used to analyze the risk factors for VTE and bleeding in patients receiving anticoagulant therapy. RESULTS: Of 205 patients, during the anticoagulant treatment, 14 patients developed DVT, and 4 patients with PE (2 of them were combined with DVT) with an incidence of 7.8% (16/205). There were 2 patients suffering from cerebral hemorrhage, 2 patients with gastric bleeding, and 1 patient with intra-tracheal hemorrhage with an incidence of 2.4% (5/205). Compared with the patients without VTE or bleeding, the R value of TEG in patients with VTE was significantly lowered (minutes: 4.6±2.2 vs. 7.4±1.4, P < 0.01), which was significantly increased in patients with hemorrhagic complications (minutes: 12.1±1.1 vs. 7.4±1.4, P < 0.01). There was no significant difference in the K value, α angle, MA value of TEG, or PT, APTT, D-dimer between the patients with and without VTE or bleeding. Multivariate Logistic regression analysis revealed that the R value of TEG was independent risk factor for incidence of VTE and hemorrhagic complication in SICU patients who receiving anticoagulation therapy [VTE: ß = 0.386, odds ratio (OR) = 1.096, 95% confidence interval (95%CI) = 1.021-2.361, P = 0.006; hemorrhagic complication: ß = -1.213, OR = 1.051, 95%CI = 1.017-3.458, P = 0.045]. CONCLUSIONS: The R value of TEG is associated with the occurrence of VTE and hemorrhagic complications in patients receiving anticoagulant therapy in SICU.

Influence of Ligand Backbone Structure and Connectivity on the Properties of Phosphine-Sulfonate Pd(II)/Ni(II) Catalysts.

Phosphine-sulfonate based palladium and nickel catalysts have been extensively studied in ethylene polymerization and copolymerization reactions. Previously, the majority of the research works focused on the modifications of the substituents on the phosphorous atom. In this contribution, we systematically demonstrated that the change of the ligand backbone from benzene to naphthalene could greatly improve the properties of this class of catalysts. In the palladium system, this change could increase catalyst stability and polyethylene molecular weights. In the nickel system, this change could dramatically increase the polyethylene molecular weights. Most interestingly, the change in the connectivity of phosphine and sulfonate moieties to the naphthalene backbone could also significantly influence the catalyst properties.

Association between polymorphisms in PDCD1 gene and aplastic anemia in Chinese Han population.

Single nucleotide polymorphism (SNP) of programmed cell death 1 (PD-1, encoded by PDCD1) has been reported to be associated with several autoimmune diseases including rheumatoid arthritis (RA), Graves' disease and multiple sclerosis (MS). In order to study the correlation between PD-1 gene polymorphism and aplastic anemia in a Chinese Han population, two SNPs, PD-1.1 G/A (rs36084323) and PD-1.6 G/A (rs10204525), were genotyped in 166 patients with aplastic anemia and 144 healthy controls by direct sequencing. All genotype distributions in both patients and controls were in Hardy-Weinberg equilibrium. Associations of genotypes and alleles with aplastic anemia were analyzed. The results suggested that the G allele of PD-1.1 was associated with an increased risk for aplastic anemia, while SNP of PD-1.6 was not associated with aplastic anemia in a Chinese Han population.

[The effects of activated protein C on the von Willebrand factor and von Willebrand factor cleaving protease of rat aortic endothelial cell induced by lipopolysaccharide].

OBJECTIVE: To investigate the activated protein C (APC) on the von Willebrand factor antigen (vWFAg) and von Willebrand factor cleaving protease (ADAMTS-13) protein expression in rat aortic endothelial cells (RAECs) induced by lipopolysaccharide (LPS). METHODS: RAECs from Wistar rats were cultured with the tissue explants adherence method. RAECs were cultured for one week, After one week culture, RAECs in 4-5 generations were divided into control group, LPS stimulation groups (1 mg/L) and APC intervention groups (0.1, 1 and 10 mg/L APC was added after LPS stimulation). The supernatants were obtained at 12, 24, 48, and 72 hours after LPS stimulated to determine the vWFAg and protein of ADAMTS-13 expression by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: In the control group, RAECs expressed little vWFAg and protein of ADAMTS-13. With stimulation of LPS, the vWFAg was significantly increased at 12 hours, and reached the peak at 48 hours [(285.45±30.13)%], and the level of ADAMTS-13 (µg/L) was gradually decreased, and reached the nadir at 72 hours (13.32±2.37), there was significant difference compared with control group [vWFAg: (94.53±7.83)%, ADAMTS-13: 115.76±2.36, both P<0.01). The effects on vWFAg promoting and ADAMTS-13 inhibition after LPS stimulation could be dose-dependently reversed by APC. 10 mg/L of APC could decrease the peak of vWFAg at 48 hours of LPS stimulation [(198.43±17.92)% vs. (285.45±30.13)%], and increase the minimize of ADAMTS-13 (µg/L) at 72 hours of LPS stimulation (125.25±2.70 vs. 13.32±2.37), with significant difference (both P<0.01). CONCLUSIONS: After stimulation with LPS, the level of vWFAg was time-dependent increased, as the protein of ADAMTS-13 was decreased. APC could attenuate the effect of LPS on vWFAg and protein of ADAMTS-13 with dose-dependent and time-dependent patterns.

[Induction of expression of von Willebrand factor cleaving protease of rat aortic endothelial cell by lipopolysaccharide challenge].

OBJECTIVE: To investigate the dose response effect and time response effect of lipopolysaccharide(LPS) on von Willebrand factor cleaving protease (ADAMTS-13, Vwf-cp) mRNA expression and protein in rat aortic endothelial cells (RAECs). METHODS: RAECs were grown by culturing of aortic tissue. When ARECs were cultured for one week, it was co-cultured by 1:3 to reach 4-5 generations. ARECs were randomly divided into five groups: control group and four LPS stimulation groups (0.01, 0.1, 1 and 5 µg/ml) . The RAECs and supernatants were obtained at 12, 24, 48, and 72 hours after being stimulated by LPS. ADAMTS-13 mRNA expression of RAECs was assessed by quantitation reverse transcription-polymerase chain reaction (RT-PCR), and protein of ADAMTS-13 in supernatants was determined by enzyme linked immunoadsorbent assay (ELISA). RESULTS: In the control group RAECs were shown to express ADAMTS-13 at both protein and mRNA levels. With the increase of concentration of LPS, or increase in stimulus duration, expression of ADAMTS-13 mRNA and protein were gradually lowered. Compared with the control group (25.22 ±1.41), the level of ADAMTS-13 mRNA in 0.01 µg/ml LPS stimulation group was markedly decreased at 48 hours (18.78±0.86, P<0.01). At 24 hours, the levels of ADAMTS-13 mRNA (23.43±0.63, 22.41±0.76) were markedly decreased in 0.1 µg/ml and 1 µg/ml LPS stimulation groups (P<0.05 and P<0.01). The level of ADAMTS-13 mRNA (20.01±2.47) in 5 µg/ml LPS stimulation group was markedly decreased at 12 hours (P<0.01). Compared with the control group [(115.76±2.36) ng/ml], protein level of ADAMTS-13 [(113.43±1.07) ng/ml] was markedly decreased at 12 hours in 0.01 µg/ml LPS stimulation group (P<0.05). The protein level of ADAMTS-13 [(7.63±2.64) ng/ml] was lowest in 5 µg/ml LPS stimulation group at 72 hours (P<0.01). CONCLUSION: Normal RAECs can express ADAMTS-13 at both mRNA and protein to certain extent. The expression of ADAMTS-13 mRNA and protein are decreased after LPS challenge in different concentrations for different duration in dose dependent and time dependent manner.

[Effect of Xuebijing injection on expression of endothelial protein C receptor and protease activated receptor 1 mRNA and protein in endothelial cells induced by lipopolysaccharide].

OBJECTIVE: To investigate the effect of Xuebijing injection on expression of endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1) mRNA and protein in rat aortic endothelial cells (RAECs) after lipopolysaccharide (LPS) challenge. METHODS: RAECs were cultured for one week, and the purity was determined with flow cytometry. ARECs were randomly divided into four groups: control group, LPS stimulation group (1 mg/L LPS), Xuebijing injection treatment group (LPS: 1 mg/L, Xuebijing injection ; 10 g/L) and activated protein C (APC) treatment group (LPS: 1 mg/L, APC: 0.1 mg/L). The RAECs were collected at 12, 24, 48, and 72 hours after being stimulated by LPS. EPCR and PAR1 mRNA of RAECs were assessed by reverse transcription-polymerase chain reaction (RT-PCR). EPCR and PAR1 protein were assessed by flow cytometry. RESULTS: At 12 hours, EPCR and PAR1 protein expressions were not significantly different among groups (all P>0.05). The level of EPCR and PAR1 mRNA were decreased in LPS stimulation group, but they were elevated in both APC and Xuebijing injection treatment groups (P<0.05 or P<0.01). From 24 to 72 hours, compared to control group, the levels of EPCR mRNA and protein expression were significantly decreased after LPS stimulation (P<0.05 or P<0.01). The levels of EPCR expression were decreased and negatively correlated with the time of LPS treatment. Also, compared to LPS stimulation group, treatment with Xuebijing markedly elevated the levels of EPCR (P<0.01). The levels of PAR1 expression were significantly decreased by LPS stimulation compared with those of control group (P<0.05 or P<0.01). After the treatment with Xuebijng, the expression of PAR1 was gradually increased (all P<0.01). Compared with APC treatment group, Xuebijing could increase the PAR1 expression better. CONCLUSION: Xuebijing could raise EPCR and PAR1 mRNA and protein expression of RAECs after LPS challenge, and it may be related to its protection of endothelial cell from undergoing apoptosis.

[Effects of Xuebijing injection on protein C and tumor necrosis factor-alpha mRNA in rats with sepsis].

OBJECTIVE: To investigate the effects of the integrated traditional Chinese medicine Xuebijing injection on protein C (PC) and tumor necrosis factor-alpha (TNF-alpha) mRNA in rats with sepsis. METHODS: Sepsis was induced in Wistar rats by cecal ligation and puncture (CLP). Ninety-six healthy animals were randomly divided into four groups: normal group, sham-operation group, CLP model group, and Xuebijing-treated group. The two latter groups were divided into 2, 8, 24, 48, and 72-hour subgroups with 8 rats in each subgroup. Platelet count of blood obtained from abdominal aorta was determined and tissue samples from liver and lungs were collected to measure tissue PC and TNF-alpha mRNA expression. RESULTS: PC gene expression levels in lung tissues were significantly lowered (all P<0.01), but they were dramatically raised by Xuebijing injection during 8-72 hours post-CLP (all P<0.01). Compared with normal group, TNF-alpha mRNA levels in liver and lungs were significantly elevated at 2 hours post-CLP (P<0.05 or P<0.01). However, treatment with Xuebijing injection markedly reduced TNF-alpha mRNA both in liver and lungs at 2-24 hours (P<0.05 or P<0.01). In CLP group, blood platelet count was significantly decreased to certain extent at different intervals within 8-72 hours, and it was markedly elevated in the Xuebijing-treated group (P<0.05 or P<0.01). CONCLUSION: The current study suggests that Xuebijing injection could exert preventing effect on the development of severe sepsis by suppressing PC and TNF-alpha mRNA.

[Effects of Xuebijing injection on thrombomodulin and endothelial cell protein C receptor in septic rats].

OBJECTIVE: To investigate effects of the integrated traditional Chinese medicine Xuebijing injection on thrombomodulin (TM) and endothelial cell protein C receptor (EPCR) in septic rats. METHODS: Ninety-six healthy Wistar rats were randomly divided into four groups: control group, sham operation group, cecal ligation and puncture (CLP) model group, and Xuebijing-treated group. Sepsis was reproduced by CLP. The two latter groups were divided into five subgroups of 2, 8, 24, 48 and 72-hour with 8 rats in each subgroup. Tissue samples from liver and lung were collected to determine tissue TM and EPCR mRNA expression. RESULTS: TM and EPCR mRNA expressions were observed in liver and lung in control group and sham operation group, while with no significant differences at 2 hours post-CLP (both P>0.05). TM and EPCR gene expression levels in tissues were significantly increased to certain extent at 8-48 hours (all P<0.01), and were dramatically decreased following Xuebijing injection at 72 hours post-CLP (both P>0.05). Also, treatment with Xuebijing injection markedly decreased TM and EPCR mRNA levels to certain extents at 8 and 24 hours, and markedly increased at 48 and 72 hours compared with those of model group. CONCLUSION: These data suggest that Xuebijing injection could raise TM and EPCR mRNA expression, thereby it might be effective in prevention of development of severe sepsis.